The methods for determining the presence or concentration of ligands or biological substances in biological fluids, by immunological means, are at present well known; they are based on the formation of a complex between the substance to be determined and one or more antibodies, it being possible for one of the components of the complex to be labelled especially by an enzyme, in order to permit its detection and/or its quantitative analysis after separation of the complexed labelled antibody or antigen from the uncomplexed labelled antibody or antigen.
The principle of the RIA and ELISA methods is also well known.
The ELISA technique (enzyme-linked immunosorbent assay) in particular is an immunoenzymatic method of very great sensitivity, for assaying of antibodies, for example, which consists in using an immunoadsorbent complex made up in particular of an antigen fixed on a solid support, for taking up the specific antibodies contained in the biological medium to be tested, such as serum, the immune complex thus obtained being detected by an anti-species antibody labelled by an enzyme, after which a substrate specific to the enzyme is added, whose degradation by the enzyme causes a coloured substance to appear; the strength of the colouring is proportional to the quantity of enzyme which reacts and, thus, to the antibody quantity present in the tested biological medium; the strength of the colouring can be assessed by the naked eye or measured by any suitable means, in particular by photometry.
In the above immunological methods using a heterogenous phase, the nature of this solid phase which serves as a support for the immunological reaction is very important since it determines the sensitivity of the measurement.
The supports which have been proposed for the ELISA test are, in particular, polyacrylamide beads activated by glutaraldehyde (AVRAMEAS and TERNYNCK, Immunochemistry, 1969, 6, pages 53-65); particles of cellulose activated by cyanogen bromide, on which antibodies are fixed by covalent bonds (ENGVALL and PERLMANN, Immunochemistry, 1971, 8, 871-874); polystyrene tubes on the surface of which antigens are fixed by simple physical adsorption (ENGVALL and PERLMANN, J. Immun. 1972, 160, 129-136); discs of paper activated by cyanogen bromide and on which an antibody or an antigen is fixed (BRIGHTON et al. Scand. 1974, 29, 166-174). However, the supports most widely used at present are the surfaces made of plastic material which are offered in the form of microplates with 96 flat-bottomed, U-shaped or V-shaped wells, of polystyrene or PVC, or small bars with 8 wells.
Another support, made up of magnetic balls, also called magnetic beads or magnetic particles, has also been described and may permit the automation of the methods used.
Mention may be made in particular of the magnetic balls described in European Patent 38,730 RHONE POULENC SPECIALITES CHIMIQUES, which describes magnetic polymer latexes, the magnetic balls described in European Patent 125,995 ADVANCED MAGNETICS INC., the magnetic balls described in French Patents 2,262,805 and 2,454,098 CORNING GLASS WORKS or the balls described in European Patent Applications SERONO DIAGNOSTICS PARTNERS 105,714, 190,006, 238,353 and 249,357.
The different magnetic balls described in the abovementioned documents can also be applied for the implementation of an immunoassay such as ELISA or RIA.
Both the RIA method and the ELISA method can be used in several ways:
The indirect method is the simplest for assaying antibodies. The antigen is fixed on the microplate (in the presence of albumin in order to block the unoccupied sites). The serum to be assayed is added and the binding of the antibodies is detected by the addition of anti-Ig antibodies suitably labelled, in particular linked to an enzyme and visualized quantitatively by spectrophotometry after addition of the substrate of the enzyme.
A competitive technique can be used for assaying the antigens. As in the preceding technique, it is the antigen which is always fixed on the plates. A mixture of antigen and of small quantities of antibody is added. The greater the number of antibody molecules neutralised by the antigen molecules, the smaller the antibody quantity fixed on the plate.
Sandwich methods can be used for assaying antigens or antibodies. For assaying antigens, the microplates are covered with the antibody, the antigen is added, then the specific antibody labelled in a suitable manner, in particular linked to an enzyme, is applied and visualized by addition of the substrate. However, the technique is applicable only to those antigens which are divalent or have several determinants. For assaying antibodies, the microplates are in contrast covered with the antigen, and the antibody to be assayed and the antigen labelled by an enzyme are added successively.
However, such methods cannot be completely automated since they do not permit an automatic assay of at least one substance in a plurality of samples. Indeed, the implementation of these different methods only permits assays of the same type in series (competitive or sandwich or immunocapture) parameter by parameter, either continuously or discontinuously, at slow speeds.